Distinct biological activity of Lewy body α-Synuclein strain in mice

Extraction of α-Synuclein (αSyn) aggregates from Lewy body disease (LBD) brains has been widely described yet templated fibrillization of LB-αSyn often fails to propagate its structural and functional properties. We recently demonstrated that aggregates amplified from LB-αSyn (ampLB) show distinct biological activities in vitro compared to human αSyn preformed fibrils (hPFF) formed de novo. Here we compare the in vivo biological activities of hPFF and ampLB regarding seeding activity, latency in inducing pathology, distribution of pathology, inclusion morphology, and cell-type preference. Injection of ampLB into mice expressing only human αSyn (Thy1:SNCA/Snca−/− mice) induced pathologies similar to those of LBD subjects that were distinct from those induced by hPFF-injection or developing spontaneously with aging. Importantly, αSyn aggregates in ampLB-injected Thy1:SNCA/Snca−/− mice maintained the unique biological and conformational features of original LB-αSyn. These results indicate that ampLB-injection, rather than conventional PFF-injection or αSyn overexpression, faithfully models key aspects of LBD.


Introduction
Lewy bodies (LB) are intraneuronal inclusions largely composed of α-Synuclein (αSyn) and are found in Lewy body diseases (LBD), a family which encompasses Parkinson's disease (PD), dementia with Lewy bodies (DLB), and Alzheimer's disease (AD) with LB co-pathology 1,2 . Patients with PD rst show motor dysfunction (i.e., parkinsonism) but eventually develop cognitive dysfunction, diagnosed as PD with dementia (PDD) 3 . A diagnosis of DLB is given to patients in whom cognitive dysfunction occurs before or concurrently with the motor dysfunction 4 . LB co-pathology is also commonly observed in subjects pathologically diagnosed with AD 2,5 . An additional synucleinopathy is represented by multiple system atrophy (MSA), a clinically distinct entity characterized by the abundance of oligodendroglial αSyn pathology 6, 7 .
αSyn is highly expressed in neurons, existing in an equilibrium of soluble cytosolic and membrane-bound α-helical forms 8 . However, recombinant αSyn monomers easily undergo aggregation in physiological buffer conditions and form β-sheet-rich amyloid structures that resemble those observed in LB in diseased brains 9,10 . These αSyn aggregates generated de novo from recombinant αSyn under controlled conditions (commonly referred to as preformed brils; PFF) have been widely used to study different aspects of LBD over the past decade. PFF work as a template to recruit and seed the misfolding of endogenous αSyn in cultured cells and neurons 11,12 . Similarly, PFF inoculation induces αSyn inclusions in animal brains, and is accompanied subsequently by intercellular transmission of the αSyn pathology 13, 14 , a phenomenon also inferred from autopsy studies on subjects with PD [15][16][17] .
Despite the clear in uence of bril structure on αSyn in vitro and in vivo pathological activity with respect to seeding potency, inclusion morphology, cell tropism, and biophysical signatures, attempts to generate recombinant brils that faithfully recapitulate the properties of brain-derived αSyn aggregates have been met with limited success. Furthermore, even though brain-derived αSyn aggregates can template the brillization of recombinant αSyn, as observed in seeded aggregation assays or protein misfolding cyclic ampli cation-based strategies using brain lysates or cerebrospinal uid, brils generated in this manner remain conformationally and functionally distinct from brain-derived αSyn aggregates 27-31 .
Highlighting these differences, we recently demonstrated differences in pathological activity in vitro and conformational features between hPFF and αSyn aggregates extracted from LBD brains (LB-αSyn) 32 . In order to bridge this gap and to overcome the limited availability and low seeding activity of LB-αSyn compared to αSyn aggregates extracted from MSA brains 20 , we established a method for amplifying LB-αSyn using recombinant human αSyn monomers. By optimizing the stoichiometry of LB-αSyn and recombinant αSyn monomers in assembly mixtures, this template-seeded ampli cation approach successfully replicated the conformational features of original LB-αSyn and unique pathological activity in vitro 32 .
Here, we aimed to uncover whether or not the distinct pathological features induced by the LB-αSyn strain are preserved in the brain. Our ndings demonstrate conspicuous differences in pathological features induced by hPFF vs ampLB in the brains of wild-type (WT) mice. We then developed a novel LBD mouse model through injection of ampli ed LB (ampLB) into mice expressing only human αSyn (Thy1:SNCA/Snca -/mice). The comparisons of the ampLB-injected Thy1:SNCA/Snca -/mice with LBD subjects, hPFF-injected Thy1:SNCA/Snca -/mice, and old Thy1:SNCA/Snca -/mice with spontaneous αSyn pathology further solidify the role of conformation as a determinant of αSyn pathological activity.
Our results provide a novel approach for improving currently available LBD models induced by PFFinjection or αSyn overexpression.

Lewy body ampli cation increases αSyn pathology induced in WT mice
We previously observed that striatal injection of LB-αSyn only induced sparse αSyn pathology when injected into WT mouse brains 20 . We therefore sought to augment the level of αSyn pathology by increasing the amount of LB-αSyn injected and by injecting ampLB ( Figure 1A). Our LB-αSyn ampli cation methodology increases the αSyn concentration 20 times, up to 200 ng/µl 32 . We injected LB-αSyn at two doses: 50 ng, the same dose as we injected previously 20 or 170 ng, the maximum dose achievable with brain-derived lysate (Table S2). For ampLB-injection, we injected either ampLB. We analyzed αSyn pathology at 3, 6, and 9 months post-injection (MPI) and quanti ed the number of phospho-serine129 αSyn (pSyn)-positive neuronal somatic inclusions. Both LB-αSyn and ampLB induced αSyn pathology in a dose-and time-dependent manner, though the higher concentration (170 ng) of LB-αSyn only induced mild αSyn pathology throughout the brain ( Figures 1B-D). Meanwhile, ampLB induced more αSyn pathology than LB-αSyn, and 500 ng of ampLB induced the most severe αSyn pathology ( Figures 1B-D). Based on these results, we decided to inject 500 ng of ampLB in mice for most experiments using WT mice.
LB-αSyn only comprises less than 2% of total protein in LBD brain lysates (Tables S2). To examine the effects of contaminants on αSyn pathogenesis in mouse primary neurons and mouse brains, we immunodepleted αSyn in LBD brain lysate ( Figure 1E). hPFF and immunodepleted brain lysate mixed with hPFF induced neurite-dominant αSyn pathology, while LB-αSyn and ampLB induced soma-dominant αSyn pathology in mouse primary neurons ( Figure 1F). For mouse brain injection, we used the following materials: hPFF, immunodepleted brain lysate mixed with hPFF (designated as mixed material), original LB-αSyn used to generate ampLB, and ampLB. hPFF-and mixed material-injected WT mice showed similar distribution and amount of αSyn pathology, while ampLB-injected samples induced signi cantly higher αSyn pathology than them at 6MPI ( Figure 1G). These results suggest that contaminants contained in LB-αSyn have minimal effects on hPFF-induced αSyn pathology both in vitro and in vivo. Moreover, the results also suggest distinct pathological activity between hPFFs and ampLBs in the presence of similar contaminants, further supporting the faithful ampli cation of LB-αSyn.

Distinct biological activity between hPFF and ampLB in WT mice
To further characterize the pathological activity of ampLB in WT mice, we generated brain lysates from 2 AD cases, 2 PDD cases, and 2 DLB cases, and determined possible differences among ampLB generated from different LBD cases. We then validated ampLB from each brain lysate by testing its ability to induce distinct morphology of αSyn pathology in mouse primary neurons ( Figure S1). We then injected WT mice with 500 ng of ampLB preparations as well as 500 ng and 5 µg of hPFF, and analyzed them histologically at 3, 6, and 9MPI ( Figure 2A). hPFF-injected samples showed the highest amount of αSyn pathology in brains at 6MPI. In contrast, all the ampLB-injected samples showed little αSyn pathology at 3MPI but showed much more pathology at 6MPI, which was further increased at 9MPI . Based on the numbers of neuronal somatic inclusions at 6MPI, ampLB preparations showed 5-50 times more seeding activity than hPFF. Compared with the numbers of neuronal inclusions at 6MPI, hPFF and ampLB preparations induced 60-80% and 5-10% of the inclusions at 3MPI, respectively, suggesting that ampLB take longer to induce αSyn pathology than hPFF ( Figure 2D). The numbers of neuronal inclusions induced by ampLB-injection in the SNpc also jumped at 6MPI but were decreased at 9MPI (Figures S2A and S2B). The numbers of tyrosine hydroxylase (TH)-positive neurons in the ipsilateral SNpc were signi cantly decreased compared with those in the contralateral SNpc in AD2 and PDD1 ampLB-injected samples at 9MPI ( Figure S2C), suggesting that the decrease in numbers of neuronal inclusions was caused by TH-positive neuron loss. Biochemical analysis revealed that injected ampLB, which were generated from human αSyn monomers, were almost completely degraded by 3MPI ( Figure S3). Meanwhile, mouse αSyn and pSyn, including their monomeric and oligomeric forms, were increased over time, suggesting that pSyn-positive pathology induced by ampLB-injection was composed of endogenous mouse αSyn.
Since we observed ~10-fold differences in seeding activity in WT mouse brains among ampLB preparations, we sought to identify the main contributing factors. One of each ampLB preparation generated from AD and PDD brain lysate (AD2 and PDD1) showed high seeding activity, suggesting that the differences in seeding activity were not disease-speci c but case-speci c. We did not nd any clear correlation between seeding activity and age at onset or disease duration (Tables S1 and S2). One factor we found was the total protein in ampLB preparations, which was negatively correlated with the seeding activity both in vivo and in vitro, albeit not reaching statistical signi cance ( Figures S4A and S4B). The in vivo and in vitro seeding activity seemed to be positively correlated (Figures S4C).
We next analyzed the distribution of αSyn pathology in hPFF-and ampLB-injected samples at 6 and 9MPI. hPFF-injected samples showed the most severe pathology in the striatum, the injection site, while all the ampLB-injected samples showed the most severe pathology in some cortical areas and the amygdala ( Figures 2E and S5A). We quantitatively measured proportion of pSyn-positive area based on the classi cation of brain regions shown in Figure S5B. Heatmap and principal component analysis showed the differences in distribution of αSyn pathology between hPFF-and ampLB-injected samples (Figures 2F,2G,S5C,and S5D). We further classi ed brain regions into several brain systems to statistically analyze the differences in distribution of αSyn pathology induced by hPFF and LBD (including AD, PDD, and DLB) ampLB preparations. The signi cant differences observed in some brain systems further validated the difference in distribution of αSyn pathology between hPFF-and ampLBinjected samples ( Figures 2H and S5E). Importantly, we did not observe clear differences among LBD (AD, PDD, and DLB) from these analyses.
Next, we examined the morphology of pSyn-positive neuronal inclusions induced by hPFF, ampLB, and LB-αSyn injection. hPFF-injected samples showed various morphology of neuronal inclusions, while ampLB-and LB-αSyn-injected samples mostly showed diffuse somatic pathology (Figures 3A. We classi ed neuronal inclusions into three types based on their morphology: diffuse somatic pathology (diffuse), isolated compact pathology (compact), and granular pathology (granule) (Figures 3B). The proportion of each morphology seen in hPFF-injected samples were clearly different from those seen in ampLB-and LB-αSyn-injected samples. All the ampLB-injected samples showed mostly diffuse pathology, and the proportion of this pathology was signi cantly different from that of hPFF-injected samples (Figures 3C). Aside from morphological differences, we found that ~10% of neurons with pSynpositive somatic inclusions also contained intranuclear inclusions in hPFF-injected mice, while these were rarely observed in ampLB-injected ones (Figures 3D) and further veri ed by confocal microscopy that a subset of neuronal inclusions in hPFF-injected samples are intranuclear.
Additionally, we found cell-type preference as another difference between hPFF-and ampLB-induced pathology. hPFF induced glial inclusions in WT mouse brains, especially in the corpus callosum, in a dose-and time-dependent manner, the observation we previously reported for mouse αSyn PFF 33 (Figures 3E and 3F). We further con rmed that these glial inclusions were present in Olig2-positive oligodendroglia, but not in glial brillary acidic protein (GFAP)-positive astrocytes or ionized calciumbinding adaptor protein-1 (Iba1)-positive microglia (Figures 3G). Signi cantly, glial inclusions were rarely observed in all the ampLB-injected samples up to 9 MPI (Figures 3I).
Modeling LBD in Thy1:SNCA/Snca -/mice Because of the signi cant differences in biological activity between hPFF and the LB-αSyn strains in cultured cells and WT mouse brains, we sought to generate a novel LBD animal model that would recapitulate the spread of the LB-αSyn strain in brain. Considering possible differences in pathology induced by human vs mouse αSyn, we generated mice that only express human αSyn by crossing mice expressing human αSyn under a mouse Thy1 promoter (Thy1:SNCA mice) with Snca knock-out (Snca -/-) mice ( Figure 4A). The resulting Thy1:SNCA/Snca -/mice expressed high levels of αSyn compared with WT mice especially in the brainstem, cerebellum, and spinal cord ( Figure S6A). However, Thy1:SNCA/Snca -/mice expressed low levels of αSyn in the SN, including dopaminergic neurons (data not shown). Western blot analysis showed Thy1:SNCA/Snca -/mice expressed ~ 4-fold more αSyn than WT mice in the entire brains ( Figure S6B). We injected 1 ug of ampLB into the dorsal hippocampus of Thy1:SNCA/Snca -/mice and conducted pathological analyses at 3, 6, and 9MPI ( Figure 4A and S6C). These mice showed severe αSyn pathology in the ipsilateral ventral dentate gyrus (DG) but only showed little pathology in other brain regions at 3MPI (Figures 4B and 4C). They showed very severe pathology in the ipsilateral hippocampus and severe pathology in some brain regions at 6MPI, with more pathology especially in the brainstem at 9MPI. Although Thy1:SNCA/Snca -/mouse neurons showed stronger pSyn immunoreactivity than WT mouse neurons, ampLB-induced αSyn inclusions were clearly distinguished based on their even stronger pSyn staining intensity and morphology. We evaluated pathological changes in the ipsilateral ventral DG because of its remarkable αSyn pathology during the time course ( Figure 4D). The number of NeuN-positive neurons in ampLB injected animals was signi cantly decreased compared with that of PBS-injected animals from 3MPI, and that was further decreased in a time-dependent manner. Both GFAP-and Iba1-positive areas were signi cantly increased compared with PBS-injected samples from 3MPI, suggesting reactive astrogliosis and microglial activation.
We conducted behavioral analyses on ampLB-injected Thy1:SNCA/Snca -/mice together with PBSinjected WT mice and Thy1:SNCA/Snca -/mice between 6 and 8MPI. The open eld test showed longer total distance traveled in Thy1:SNCA/Snca -/mice than that of PBS-injected WT mice, indicative of their hyperactivity ( Figure S6D). The Y maze test showed no signi cant differences in alternation among the groups ( Figure S6E). In the probe trial of the Barnes maze, time spent in target zone was not different between day1 and day10 in PBS-injected Thy1:SNCA/Snca -/mice, while that was signi cantly decreased in ampLB-injected Thy1:SNCA/Snca -/mice at day10 compared with day1 ( Figures 4E and  S6F). Likewise, in the cued fear conditioning test, freezing time during auditory cue was not different between day1 and day10 in PBS-injected Thy1:SNCA/Snca -/mice, while that was signi cantly decreased in ampLB-injected Thy1:SNCA/Snca -/mice at day10 compared with day1 ( Figures 4F and  S6G). The time difference between day1 and day10 was signi cantly decreased in ampLB-injected Thy1:SNCA/Snca -/mice compared with PBS-injected Thy1:SNCA/Snca -/mice. These results suggest that injection of ampLB into the hippocampus induced impairment of spatial and cued fear memory retention.
Pathological and phenotypic features of hPFF-injected Thy1:SNCA/Snca -/mice and old Thy1:SNCA/Snca -/mice with spontaneous αSyn pathology Aside from ampLB-injection, we also applied hPFF-injection to Thy1:SNCA/Snca -/mice ( Figure 5A). hPFF-injected Thy1:SNCA/Snca -/mice showed rapid spread of αSyn pathology in the brain with very severe pathology in the ipsilateral DG and severe pathology in the brainstem regions at 3MPI, followed by very severe pathology in the ipsilateral DG, brainstem, and spinal cord at 6MPI ( Figures 5C and 5D). Mice showed paralysis and ataxia from ~5MPI and did not survive beyond 6MPI ( Figure 5B and Movie S1).
We found that some of the old Thy1:SNCA/Snca -/mice without ampLB-or hPFF-injection developed spontaneous αSyn pathology over 13 months of age. We rst observed that some Thy1:SNCA/Snca -/mice exhibited paralysis and ataxia and then died ( Figure 5E). Their motor dysfunction resembled that of hPFF-injected mice. All of the mice showed very severe pathology especially in the brainstem and spinal cord (Figures 5F and 5G). We also sacri ced old asymptomatic Thy1:SNCA/Snca -/mice and found less severe pathology in some of them. We classi ed them into mildly and moderately affected cases based on the severity of αSyn pathology (Figures 5E-G).
In order to determine whether conformational properties of ampLB continued to be propagated in vivo, we next conducted partial proteinase K (PK) digestion on αSyn aggregates isolated from Thy1:SNCA/Snca -/mouse brains and the original LB-αSyn used for the ampLB-injection. Digestion reactions were stopped at 1, 5, 15, and 30 min. αSyn aggregates from ampLB-injected Thy1:SNCA/Snca -/mice and the original LB-αSyn showed similar digestion pro les, which were different from those from hPFF-injected Thy1:SNCA/Snca -/mice and Thy1:SNCA/Snca -/mice with spontaneous αSyn pathology ( Figure 7C). We conducted PK digestion on multiple samples from each group for 15 min and ran them in a single gel. The samples from each group showed similar digestion pro les, and largely re ected the treatment group from which they were derived. Altogether, these results suggest that pathological αSyn aggregates in ampLB-injected Thy1:SNCA/Snca -/mice maintained the biological and conformational features of the original LB-αSyn. Moreover, these features are clearly different from those in hPFF-injected Thy1:SNCA/Snca -/mice and Thy1:SNCA/Snca -/mice with spontaneous αSyn pathology. Interestingly, the pathological αSyn aggregates in hPFF-injected Thy1:SNCA/Snca -/mice and Thy1:SNCA/Snca -/mice with spontaneous αSyn pathology showed similar biological and conformational features to each other.

Discussion
Although multiple previous studies have investigated the effect of injecting LBD brain lysates into animal brains, a unique pathological pro le induced by LB-αSyn has not been reported thus far 14,20,21,26,[34][35][36] . This may be partly due to the low αSyn yield and relatively low seeding activity of LB-αSyn compared with αSyn extracted from MSA brains. In this study, we described for the rst time the detailed biological activity of a prototypic LB-αSyn strain in animal brains by using an ampli cation methodology which we recently established 32 .
This approach allowed us to compare the pathological features induced by the same doses of hPFF and ampLB. We report here differences in biological activity between hPFF and ampLB in WT mouse brains that are re ected in seeding activity, latency in inducing pathology, distribution of pathology, morphology of neuronal inclusions, and cell-type preference. Meanwhile, we did not observe obvious disease-speci c differences in biological activity among ampLB preparations generated from AD, PDD, and DLB brain lysates. These results are consistent with a recent Cryo-EM study showing essentially undistinguishable atomic-level core structures among PD, PDD and DLB αSyn laments 28 , and suggest that the differences in clinical and pathological features among these LBD may arise from other factors than strain differences alone. However, we observed case-speci c differences of up to ~ 10-fold in seeding activity in WT mouse brains among ampLB preparations. High seeding activity is important to su ciently induce αSyn pathology and model LBD in animals. In this study, we found a negative correlation between seeding activity and total protein or contaminants in ampLB preparations, but further studies are needed to identify the factors affecting seeding activity. Nonetheless, our results showed that seeding activity in vitro may help predict the amount of pathology induced in animal brains.
Currently, PFF-injected animals are widely used for modeling and understanding LBD. However, signi cant differences in biological activity between hPFF and the LB-αSyn strain prompted us towards generating a novel LBD model employing ampLB as a pathological seed in a host that only expresses human αSyn. AmpLB-injected Thy1:SNCA/Snca -/mice recapitulated several characteristics of LBD including the presence of pSyn-positive neuronal inclusions, neuron loss, glial activation, and behavioral abnormalities. As a comparison, we also challenged Thy1:SNCA/Snca -/mice with hPFF-injection, which induced rapid spread of αSyn pathology. Interestingly, we found that some of old Thy1:SNCA/Snca -/mice developed αSyn inclusions without ampLB-or hPFF-injection, which was not reported in the original Thy1:SNCA mice 37 . The exacerbation of αSyn pathology in Thy1:SNCA/Snca -/mice compared with Thy1:SNCA mice may arise from at least two potential possibilities: (1) differences in genetic background and (2) the absence of endogenous mouse αSyn expression. The latter is consistent with previous studies showing that deletion of endogenous mouse αSyn accelerates aggregation of human αSyn overexpressed in cultured cells and in mice 38, 39 .
We examined the similarities and differences in brain pathological features and biological and conformational features of αSyn aggregates in brain lysates among the Thy1:SNCA/Snca -/mouse models and LBD subjects. AmpLB-injected Thy1:SNCA/Snca -/mice and LBD subjects showed similarities to each other, while they showed clear differences from hPFF-injected Thy1:SNCA/Snca -/mice and old Thy1:SNCA/Snca -/mice with spontaneous αSyn pathology. These results have potentially signi cant implications. First, αSyn aggregates in ampLB-injected Thy1:SNCA/Snca -/mouse brains propagate the biological and conformational features of original LB-αSyn throughout the process of ampli cation, brain injection, and in vivo incubation. This further suggests that the similarities of pathological features between ampLB-injected Thy1:SNCA/Snca -/mice and LBD subjects come from their strain similarities. However, we did not observe αSyn inclusions in ampLB-injected Thy1:SNCA/Snca -/mice that morphologically resemble the brainstem LB consisting of a central core and surrounding halo in LBD subjects. Longer incubation times may be needed for such prototypical LB to be developed, or this may be attributed to different pathological responses between mice and humans.
The other important implication is that αSyn aggregates in Thy1:SNCA/Snca -/mice with spontaneous αSyn pathology showed different features from LB-αSyn even though both Thy1:SNCA/Snca -/mice and LBD subjects spontaneously develop human αSyn aggregates. These results suggest that the LB-αSyn strain is not replicated by human αSyn overexpression in mice. In other words, introduction of the LB-αSyn strain into human αSyn-expressing mice is required to faithfully recapitulate LBD pathology. Further studies are needed to investigate what factors are required to generate the LB-αSyn strain con rmation in animals and cultured cells expressing human αSyn.
A limitation of this study is that we indirectly examined conformations of αSyn aggregates by partial PK digestion. It is important to directly examine the near-atomic core structures of LB-αSyn, ampLB, and αSyn aggregates in ampLB-injected animals by Cryo-EM in future studies. Furthermore, since previous studies have shown that brillization buffer and post-translational modi cations may affect conformations of tau laments 40, 41 , there might be room to further optimize LB-αSyn ampli cation methodology.
In conclusion, we detail the similarities and differences in features of αSyn brain pathology and pathological αSyn aggregates among mouse models and LBD subjects, highlighting the ampLB-injection as a novel strategy for improvement upon conventional PFF-injection or αSyn overexpression animal models. Considering the unique pathological mechanisms induced by the LB-αSyn strain, ampLB-injected animal models will provide new opportunities to identify therapeutic targets, develop diagnostic imaging tools, and test disease-modifying therapies for LBD.

Declarations
Data availability Source data will be shared by the lead contacts upon request. This paper does not report original code. Any additional information needed to reanalyze the data reported in this paper is available from the lead contacts upon request.

Human patient samples
Detailed clinical characteristics (disease duration, age at death, site of onset, etc.) were ascertained from an integrated neurodegenerative disease database in CNDR at the University of Pennsylvania. Frozen and para nized postmortem brain samples were obtained from patient brain donors who underwent autopsy at CNDR between 2002 and 2018. More details on these patients are found in Table S1. All procedures were performed in accordance with local institutional review board guidelines. Written informed consent for autopsy and analysis of tissue sample data was obtained either from patients themselves or their next of kin.
Biochemical extraction of sarkosyl-insoluble αSyn from human and mouse brains Biochemical brain extraction was conducted as described previously with minor modi cations 32 . All human brain tissues were obtained from the CNDR brain bank 42 . Fontal cortex tissues with a high burden of αSyn pathology from patients with AD, PDD, and DLB were identi ed by postmortem neuropathological examination 5 . Biochemical extraction of human brains was performed as described previously 32 . In brief, 5-10 g of frontal cortical gray matter was homogenized in ve volumes (w/v) of 1% (v/v) Triton X-100-containing high-salt (HS) buffer (50 mM Tris-HCl pH 7.4, 750 mM NaCl, 10 mM NaF, 5 mM ethylenediaminetetraacetic acid [EDTA]) with protease and protein phosphatase inhibitors, incubated on ice for 20 min, and centrifuged at 180,000 ´ g for 30 min. The pellets were then re-extracted with ve volumes of 1% (v/v) Triton X-100-containing HS buffer, followed by sequential extraction with ve volumes of HS buffer with 30% (w/v) sucrose for myelin oatation. The pellets were then resuspended and homogenized in 2% (w/v) sarkosyl-containing HS buffer, rotated at room temperature for 1 h or at 4 °C overnight and centrifuged at 180,000 ´ g for 30 min. The resulting sarkosyl-insoluble pellets were washed once with Dulbecco's PBS (DPBS, Corning #21-031-CV) and re-suspended in DPBS by sonication (QSonica Microson XL-2000; 60 pulses, setting 2, 0.5 s per pulse). This suspension termed the "sarkosyl-insoluble fraction" or "brain lysate" contained pathological αSyn referred to as "LB-αSyn" and was used for the experiments. Mouse brain extraction was performed with the same protocol for human brain extraction except that only one round of extraction with 1% Triton X-100-containing HS buffer was performed ( Figure S8A). The Triton X-100-soluble fraction was used for the experiments in Figure S6B.
The concentrations of αSyn in the sarkosyl-insoluble fractions were determined by sandwich ELISA (see 'Sandwich ELISA'), and the protein concentrations were examined by bicinchoninic acid (BCA) assay (Tables S2 and S3).

Sandwich ELISA
Sandwich ELISA was conducted as described previously 32 . To measure the concentration of αSyn in brain lysates, 384-well Nunc Maxisorp clear plates were coated with 100 ng (30 μl per well) of an antihuman αSyn antibody Syn9027 (CNDR) in sodium carbonate buffer, pH 9.6 and incubated overnight at 4°C . The plates were washed 4 times with PBS containing 1% (v/v) Tween 20 (PBS-T), and blocked using Block Ace solution (AbD Serotec) overnight at 4 °C. Brain lysates were sonicated with a Diagenode Biorupter sonicator (20 min, 30 s on, 30 s off, 10 °C, high setting), serially diluted in PBS and added to each well. The plates were incubated overnight at 4 °C. The recombinant human αSyn monomer and hPFF were used as standards. The plates were then washed with PBS-T and an anti-human αSyn antibody MJFR1 (Abcam #ab138501, 1:1000) or an anti-human αSyn antibody HuA (CNDR, 1:2000) was added to each well and incubated at 4 °C overnight. After washing, a secondary antibody conjugated with horse radish peroxidase (Cell Signaling Technology #7074, 1:10000) was added to the plates followed by incubation for 1 hr at 37 °C. Following another wash, the plates were developed for 10-15 min using 1- Step Ultra TMB-ELISA substrate solution (Thermo Fisher Scienti c #37574, 30 μl per well), the reaction was quenched using 10% phosphoric acid and plates were read at 450 nm on a Molecular Devices Spectramax M5 plate reader.

Recombinant αSyn puri cation and in vitro PFF generation
Puri cation of recombinant human αSyn and generation of hPFF was conducted as described previously 43 . The pRK172 plasmid containing a full-length human αSyn gene was transformed into BL21 (DE3) RIL-competent E. coli (Agilent Technologies #230245). A single colony from the transformed bacteria was expanded in Terri c Broth (12 g/l of Bacto-tryptone, 24 g/l of yeast extract 4% (v/v) glycerol, 17 mM KH 2 PO 4 and 72 mM K 2 HPO 4 ) with ampicillin. Bacterial pellets from the growth were sonicated, and the sample was boiled to precipitate undesired proteins. The supernatant was dialyzed with 10 mM Tris, pH 7.6, 50 mM NaCl, 1 mM EDTA overnight. Protein was ltered with a 0.22 μm lter and concentrated using Amicon Ultra-15 centrifugal lters (Millipore Sigma #UFC901008). Protein was then loaded onto a Superdex 200 column and 1 ml fractions were collected. Fractions were run on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and stained with Coomassie blue to select fractions that were highly enriched in αSyn. These fractions were combined and dialyzed in 10 mM Tris, pH 7.6, 50 mM NaCl, 1 mM EDTA overnight. Dialyzed fractions were applied to a HiTrap Q HP anionexchange column (GE Healthcare #17115301) and run using a linear gradient from 25 mM NaCl to 1 M NaCl. Collected fractions were run on SDS-PAGE and stained with Coomassie blue. Fractions that were highly enriched in αSyn were collected and dialyzed with DPBS. Protein was ltered through a 0.22 μm lter and concentrated to 5 mg/ml (αSyn) with Amicon Ultra-15 centrifugal lters. αSyn monomer was aliquoted and frozen at −80°C. For preparation of PFF, αSyn monomer was shaken at 1,000 rpm for 7 d.
Conversion to PFF was validated by sedimentation at 100,000 ´ g for 60 min and by Thio avin S staining.
Immunocytochemistry and quanti cation of neuron pathology Immunocytochemistry and quanti cation of pSyn-positive neuronal pathology was performed as described previously 32 . Mouse primary neurons cultured in 96 wells were washed with PBS once and xed at DIV 21 with 4% (w/v) PFA, 4% (w/v) sucrose, and 1% (v/v) Triton X-100 in PBS. After PBS washes, cells were blocked with 3% (w/v) bovine serum albumin (BSA), 5% (w/v) fetal bovine serum in DPBS for 1 h at room temperature, then incubated with an anti-pSyn antibody 81A (CNDR, 1:5000) and an antimicrotubule associated protein 2 (MAP2) antibody #17028 (CNDR, 1:3000) at 4 °C overnight. The cells were washed 5 times with PBS and incubated with secondary antibodies conjugated with Alexa uor 488 or 594 (Molecular Probes, 1:1000) for 2 h at room temperature. After washing with PBS, the cells were incubated in DAPI solution (ThermoFisher #D21490, 1:10,000 in PBS) after staining with secondary antibodies and the plates were sealed with adhesive covers. The 96 well plates were scanned with an In Cell Analyzer 2200 (GE Healthcare) with a 10 × or 40 × objective and analyzed using the accompanying software (In Cell Toolbox Analyzer). Quantitation of total 81A signal and the amount of somatic 81A signal was calculated using Cell Pro ler ver. 3.1.9 (The Broad Institute). The fraction of somatic inclusions was calculated as the 81A signal intensity (density times area) of somatic objects divided by the total 81A signal intensity. Data are reported as the average of 3 replicate wells for each treatment sample.
Immunodepletion of αSyn from brain lysate An anti-human αSyn monoclonal antibody 9027 was covalently conjugated to Dynabeads M-280, tosylactivated (Invitrogen #14204) per the manufacturer's instructions. Immunodepletion of αSyn was performed by incubating diluted AD1 brain lysate (10 ng/μl of αSyn, 50 μl total dose) with anti-αSyn antibody-bead complexes containing 68 μg of the antibody at 37 °C for 1 h with constant rotation. The immunodepleted fraction was separated from the antibody-bead complex using a magnet. Mock immunodepletion was performed using the equal amount of a control mouse IgG antibody (Jackson Immuno Research). The brain lysate immunodepleted with the anti-αSyn antibody and the control antibody were used for western blot analysis. The αSyn-depleted brain lysate (2.5 μl) mixed with hPFFs (500 ng) was used for mouse brain injection. Diluted AD1 brain lysate (10 ng/μl of αSyn, 2.5 μl total dose) and ampli ed LB-αSyn generated from AD1 brain lysate (200 ng/μl of αSyn, 2.5 μl total dose) were used for injection as comparisons. Those three injection materials contained almost the same contaminants.
Partial PK digestion of αSyn aggregates in brain lysate Sarkosyl-insoluble fractions from LBD brains and Thy1:SNCA/Snca -/mouse brains were prepared by biochemical brain extraction. For partial PK digestion, 50 ng of αSyn from each sample was sonicated with a Diagenode Biorupter sonicator (20 min, 30 s on, 30 s off, 10 °C, high setting) and mixed with 0.2 μg of PK in DPBS to a nal volume of 50 μl and incubated at 37 °C for 1, 5, 15, and 30 min. The reaction was stopped with 1 mM PMSF. The samples were boiled with SDS-sample buffer for 10 min and resolved on NuPAGE Novex 12% Bis-Tris gels (Invitrogen). Transferred nitrocellulose membranes were probed with an anti-human αSyn antibody HuA (CNDR, 1:500) and an anti-αSyn antibody Syn1 (BD transduction #610787, 1:500).
To quantify the amount of αSyn pathology, every 20 th para n section throughout the brains was stained with an anti-pSyn antibody EP1536Y, and pSyn-positive neuronal somatic inclusions with visible nuclei were manually counted. To assess distribution and severity of αSyn pathology, semi-quantitative analyses were performed for pSyn-positive pathology on the ve coronal sections (2.80, 0.26, −1.58, −2.92, and −4.04 mm relative to bregma), and color coded onto heat maps ( Figures 1C, 1G, 2E, 4C, 5D, 5G, and S5A). The extent of αSyn pathology was graded as 0-3 (0, no pathology; 0.5, mild; 1, moderate; 2, severe; 3, very severe) based on the criteria described previously 44 , and averaged across samples for each brain region. To quantitatively assess distribution of pSyn-positive area, each brain region shown in Figure S5B was measured using QuPath software 45 . The proportion of pSyn-positive area was averaged across samples for each brain region, and color coded onto heat maps (Figures 2F and S5C). Primary component analysis of distribution of pSyn-positive pathology was performed using GraphPad Prism Software, Version 9.
To assess dopaminergic neuron loss in the SNpc, every 20 th section was stained with an anti-TH antibody throughout the SNpc. The numbers of TH-positive cells with visible nuclei were manually counted. To assess neuron loss and glial activation in the ventral DG, sections at −3.52 mm relative to bregma were stained with anti-NeuN, GFAP, and Iba1 antibodies. The numbers of NeuN-positive neurons were automatically counted and the GFAP-and Iba1-positive area was measured using QuPath software.

Behavioral analysis
WT mice injected with PBS, Thy1:SNCA/Snca -/mice injected with PBS, and Thy1:SNCA/Snca -/mice injected with ampLB were subjected to behavioral tests from 6 to 8MPI. Before every test, the mice were habituated to the experimental environment for more than 30 min. Samples that encountered technical problems were removed from the analyses.

Open eld
Mice were placed at the center of the eld inside an open eld apparatus (36 × 36 cm) and allowed to move freely for 15 min. The distance traveled and time spent in the center area (18 × 18 cm) were recorded using video tracking software EthoVision XT 15 (Noldus).

Y-maze
Mice were placed at the end of one arm of the Y-maze apparatus (San Diego Instruments) and allowed to move freely for 5 min. The distance traveled and series of arm entries were recorded using video tracking software EthoVision XT 15 (Noldus). An alternation was de ned as entries into all three arms on consecutive occasions. The number of maximum alternations was therefore the total number of arm entries minus two, and the percentage of alternations was calculated.

Barnes maze
The Barnes maze test was conducted on a white circular surface with 20 holes equally spaced along the perimeter ( Figure S6F). For acquisition trials, a shelter was placed under one of the holes, i.e. the "target hole". Mice were placed in the center and allowed to move freely up to 3 min. The latency to reach the target, numbers of holes visited other than the target, and distance traveled were recorded using video tracking software EthoVision XT 15 (Noldus). Mice were subjected to acquisition trials twice a day for 7 d, and then to probe tests 24 h and 10 d after the last acquisition trial. In the probe tests without the shelter, mice were placed in the center and allowed to move freely for 3 min. The time spent around each hole, numbers of visiting each hole, and distance traveled were recorded. "Target zone" were de ned as the target hole plus the 2 holes on either side of the target hole.

Contextual fear conditioning
Conditioning and test sessions were performed in a standard operant chamber (Med Associates) equipped with a tone generator and house light. Mice were handled for habituation in front of the apparatus for 2 min per day for 3 d. On day 0, conditioning was performed ( Figure S6G). Mice were placed in a test chamber inside a sound-attenuated cabinet and allowed to explore freely for 150 s. A white noise, which is conditioned stimulus, was presented for 30 s, followed by a foot shock (2 s, 1 mA) serving as unconditioned stimulus. On day 1 and 10, contextual fear memory and auditory-cued fear memory tests were performed. For the contextual fear memory test, mice were placed in the same chamber in the same context as the conditioning, and immobile time and distance traveled were recorded for 5 min. For the auditory-cued fear memory test, mice were placed in a different chamber in a different context from the conditioning. Mice were allowed to move freely for 2.5 min, and then the white noise was presented for 2.5 min. Immobile time and distance traveled were recorded automatically.

Quanti cation and statistical analyses
Numbers of samples or animals analyzed in each experiment, statistical analysis performed, as well as p values for all results are described in the gure legends. For all the in vivo and in vitro experiments, "n" represents the number of animals and replicates, respectively. An F test, a Brown-Forsythe test, or a Bartlett's test was performed to evaluate the differences in variances. An unpaired, two-tailed Student's ttest, a two-tailed paired test, or a Mann-Whitney test was used to determine statistical signi cance between two groups. One-or two-way Analysis of Variance (ANOVA) with a Dunnett's, Tukey's, or Sidak's multiple comparison test was used to determine statistical signi cance among three or more groups. A linear regression model was used to test the correlation between two variables. A Fisher's exact test was Page 22/34 used to analyze contingency table data. Statistical calculations were performed with GraphPad Prism Software, Version 9. Differences with p values of less than 0.05 were considered signi cant. Statistically signi cant comparisons in each gure are indicated with asterisks, *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. Data are presented as mean ± SEM. analysis of AD1 brain lysate immunodepleted with a control mouse IgG or an anti-human αSyn antibody 9027. Immunoblots with anti-human αSyn (HuA) and pSyn (81A) antibodies. (F) Left panels: Mouse primary hippocampal neurons treated with hPFF, immunodepleted (ID) AD1 brain lysate mixed with hPFF, LB-αSyn (AD1), and ampLB (AD1). Note that the ID brain lysate mixed with hPFF and LB-αSyn contain almost the same contaminants. Scale bars 100 µm, 20 µm (inset). Immunocytochemistry with anti-MAP2 and pSyn (81A) antibodies. Right panel: Percent of total pSyn-positive pathology in neuronal somatic inclusions (n = 3 per group). One-way ANOVA with a Tukey's post-hoc test was performed; **p < 0.01.(G) Upper panel: Heat map colors represent the extent of pSyn-positive pathology at 6MPI. Lower panel: Number of pSyn-positive neuronal somatic inclusions in the entire brains (n = 3-4 per group). One-way ANOVA with a Tukey's post-hoc test was performed; ****p< 0.0001. Data are represented as mean ± SEM. of diffuse, compact, and granular pSyn-positive inclusions. Lower panels: Comparison of the proportions of diffuse, compact, and granular inclusions (hPFF, n = 50; ampLB, n = 959; LB-αSyn, n = 29). A Fisher's exact test was performed between two groups for differences in the percentages of diffuse and nondiffuse inclusions; ****p< 0.0001, n.s., not signi cant. (C) Percent of diffuse inclusions in hPFF-and ampLB-injected mice (n = 3-5 per group). One-way ANOVA with a Tukey's post-hoc test was performed; ****p < 0.0001. (D) Immunohistochemistry and z-stack confocal microscopy images showing pSynpositive intranuclear inclusions in hPFF-injected mice. Scale bars 10 µm. (E) Immunohistochemistry with a pSyn antibody (EP1536Y) in the corpus callosum at 9MPI. (F) Double immuno uorescence for Olig2 (green) and pSyn (81A, red), GFAP (green) and pSyn (EP1536Y, red), and Iba1 (green) and pSyn (81A, red).
Data are represented as mean ± SEM.